The in vivo identification of a target cell population is required, and required quickly, in many industries. Such applications include those where the selected cells are destined for other applications that require the cells to be living after identification. For example, cells are processed using fluorescence-activated cell sorting, where cells are cultured and expanded in vitro after sorting, or in sperm sorting by gender in animal husbandry applications.
Being able to pre-select animal offspring gender would allow more efficient operations of livestock producers. Dairy farmers have little use for most bull calves. For example, males are preferred in beef cattle and sheep because males grow faster, producing more meat more quickly.
The male reproductive cells, the sperm, determine the gender of the offspring. Most males carry an X and a Y sex chromosome, whereas females carry two X chromosomes. A sperm or an egg contains one half of that parent's genetic information; however, the egg only carries an X chromosome one of each pair of autosomes. In mammals, the egg always contains an X chromosome, while the sperm carries either an X or Y chromosome.
Distinguishing male-producing from female-producing sperm is most easily accomplished by exploiting the difference in the size of the two sex chromosomes. The X chromosome contains more DNA than does the Y chromosome. For example, the difference in total DNA between X-bearing sperm and Y-bearing sperm is 3.4% in boar, 3.8% in bull, and 4.2% in ram sperm.
Distinguishing Cells
To illuminate the workings of cells or distinguish cells that differ from each other by the slightest difference (e.g., expression of a particular molecule), various visualization methods have been used for decades, from simple light microscopic observations to high-voltage electronic microscopy. In most of these techniques, cells or tissue are preserved, usually using a cross-linking agent such as an aldehyde (proteins, e.g., glutaraldehyde and formaldehyde), osmium (lipids) or by precipitating parts of the cells, such as cold methanol and proteins. These techniques suffer from the preparation processes that allow for the visualization. Fixation procedures often incur artifacts; for example, in the early days of electronic microscopy (EM), multilamellar bodies were observed but were later understood to be mostly by-products of the fixation protocols, not actual structures found in living mammalian cells. While fixation protocols do preserve some of the cell structure, there are many structures that are difficult to preserve, or when preserved under appropriate conditions, the rest of the cell architecture is destroyed. Classically, this has been the case for the cytoskeleton, especially for exceptionally dynamic microtubules.
To overcome the limitations of visualization techniques in fixed samples, “in vivo” approaches have been explored. For example, to understand where native polypeptides localize, those polypeptides have been purified, associated with a detectable dye (usually covalently), and then introduced into the cell of interest and observed (Chamberlain and Hahn, 2000). This approach does offer the advantages of non-fixed cells; however, the time and expense to purify a target polypeptide, conjugate it to a dye, and then to microinject (a task requiring specialized equipment, experience, skill and patience) the complex into a cell often outweigh the advantages. Furthermore, only limited numbers of cells could be examined at any given time due to the limitations of microinjection.
With the advent of the discovery of green and other visible fluorescent proteins (VFPs), however, the ability to visualize polypeptides—even polypeptide-polypeptide interactions—became facile and less riddled with artifacts. Green fluorescent protein is a naturally occurring luminescent protein first found in jellyfish. Having been cloned, many variants have been produced that produce a rainbow of colors. In most instances, the protein of interest is fused by recombinant procedures to a VFP of choice and the transgene introduced and expressed in the cell of interest (Chamberlain and Hahn, 2000). While this approach is far superior to previous methods, many extra, time-consuming, steps are required from identifying the protein of interest to actually visualizing it in a living cell.
Going beyond cellular localization and movement of proteins, other dyes have been exploited to identify other processes or stain specific molecules. For example, calcium-mediated signaling is monitored in living cells using the fura series of dyes. Other fluorescent dyes have been used to test the molecular size barriers of gap junctions in, for example, epithelial cells. Finally, other stains target specific molecules, such as double-stranded deoxyribonucleic acid (DNA); such stains include some of the Hoechst series of dyes, propidium iodide and ethidium bromide.
In each case, however, the challenge of introducing the dye or stain into a living cells to the appropriate target area is hindered by the cell membrane which provides a barrier to cells from the outside world. In many cases, dyes are membrane impermeant due to their hydrophobic nature or their size; even membrane-permeant dyes can require long incubation times to breach the membrane and reach the target molecules or cellular compartments. Breaching the barrier requires a physical perturbation of the membrane, such as by microinjection or fixation.
Available procedures are few and when available, often face uncompromising challenges. Even traditional methods of staining DNA in common methods of sorting sperm cells by gender require extensive incubation times at elevated temperatures (e.g., 60 minutes at 35° C.; (Johnson, 1992)), permitting quality degradation of the cells. In addition, staining must be sufficient so that the signal can be accurately and precisely detected.